The present invention relates to novel kinase polypeptides, nucleotide sequences encoding the novel kinase polypeptides, as well as various products and methods useful for the diagnosis and treatment of various kinase-related diseases and conditions.
The following description of the background of the invention is provided to aid in understanding the invention, but is not admitted to be or to describe prior art to the invention.
Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins, which enables regulation of the activity of mature proteins by altering their structure and function.
The best characterized protein kinases in eukaryotes phosphorylate proteins on the hydroxyl moiety of serine, threonine and tyrosine residues. These kinases largely fall into two groups, those specific for phosphorylating serines and threonines, and those specific for phosphorylating tyrosines. Some kinases, referred to as xe2x80x9cdual specificityxe2x80x9d kinases, are able to phosphorylate on tyrosine as well as serine/threonine residues.
Protein kinases can also be characterized by their location within the cell. Some kinases are transmembrane receptor-type proteins capable of directly altering their catalytic activity in response to the external environment such as the binding of a ligand. Others are non-receptor-type proteins lacking any transmembrane domain. They can be found in a variety of cellular compartments from the inner surface of the cell membrane to the nucleus.
Many kinases are involved in regulatory cascades wherein their substrates may include other kinases whose activities are regulated by their phosphorylation state. Ultimately the activity of some downstream effector is modulated by phosphorylation resulting from activation of such a pathway.
Protein kinases are one of the largest families of eukaryotic proteins with several hundred known members. These proteins share a 250-300 amino acid domain that can be subdivided into 12 distinct subdomains that comprise the common catalytic core structure. These conserved protein motifs have recently been exploited using PCR-based cloning strategies leading to a significant expansion of the known kinases.
Multiple alignment of the sequences in the catalytic domain of protein kinases and subsequent parsimony analysis permits the segregation of related kinases into distinct branches or subfamilies including: tyrosine kinases, cyclic-nucleotide-dependent kinases, calcium/calmodulin kinases, cyclin-dependent kinases and MAP-kinases, serine-threonine kinase receptors, and several other less defined subfamilies.
Through the use of a targeted PCR cloning strategy and of a xe2x80x9cmotif extractionxe2x80x9d bioinformatics script, mammalian members of the STE20-kinase family have been identified as part of the present invention. Multiple alignment and parsimony analysis of the catalytic domain of all of these STE20-family members reveals that these proteins cluster into 9 distinct subgroups. Classification in this manner has proven highly accurate not only in predicting motifs present in the remaining non-catalytic portion of each protein, but also in their regulation, substrates, and signaling pathways. The present invention includes the partial or complete sequence of new members of the STE20-family, their classification, predicted or deduced protein structure, and a strategy for elucidating their biologic and therapeutic relevance.
Thus, a first aspect of the invention features an isolated, enriched, or purified nucleic acid molecule encoding a kinase polypeptide selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5.
By xe2x80x9cisolatedxe2x80x9d in reference to nucleic acid is meant a polymer of nucleotides conjugated to each other, including DNA and RNA, that is isolated from a natural source or that is synthesized. The isolated nucleic acid of the present invention is unique in the sense that it is not found in a pure or separated state in nature. Use of the term xe2x80x9cisolatedxe2x80x9d indicates that a naturally occurring sequence has been removed from its normal cellular (i.e., chromosomal) environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only nucleotide chain present, but that it is essentially free (about 90-95% pure at least) of non-nucleotide material naturally associated with it, and thus is distinguished from isolated chromosomes.
By the use of the term xe2x80x9cenrichedxe2x80x9d in reference to nucleic acid is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased. The term xe2x80x9csignificantxe2x80x9d is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other nucleic acids of about at least 2 fold, more preferably at least 5 to 10 fold or even more. The term also does not imply that there is no DNA or RNA from other sources. The other source DNA may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector such as pUC19. This term distinguishes from naturally occurring events, such as viral infection, or tumor type growths, in which the level of one mRNA may be naturally increased relative to other species of mRNA. That is, the term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.
It is also advantageous for some purposes that a nucleotide sequence be in purified form. The term xe2x80x9cpurifiedxe2x80x9d in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation). Instead, it represents an indication that the sequence is relatively more pure than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/mL). Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones could be obtained directly from total DNA or from total RNA. The cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 106-fold purification of the native message. Thus, purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
By a xe2x80x9ckinase polypeptidexe2x80x9d is meant 32 (preferably 40, more preferably 45, most preferably 55) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or the corresponding full-length amino acid sequence; 250 (preferably 255, more preferably 260, most preferably 270) or more contiguous amino acids set forth in the amino acid sequence SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:105, or the corresponding full-length amino acid sequence; 27 (preferably 30, more preferably 40, most preferably 45) or more contiguous amino acids set forth in the amino acid sequence SEQ ID NO:18; 16 (preferably 20, more preferably 25, most preferably 35) or more contiguous amino acids set forth in the amino acid sequence SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:103 or the corresponding full-length amino acid sequence; 6 (preferably 10, more preferably 15, most preferably 25) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:97 or SEQ ID NO:99, 22 (preferably 30, more preferably 35, most preferably 45) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:101, or the corresponding full-length amino acid sequence; 78 (preferably 80, more preferably 85, most preferably 90) or more contiguous amino acids set forth in the amino acid sequence SEQ ID NO:107 or functional derivatives thereof as described herein. For sequences for which the full-length sequence is not given, the remaining sequences can be determined using methods well-known to those in the art and are intended to be included in the invention. In certain aspects, polypeptides of 100, 200, 300 or more amino acids are preferred. The kinase polypeptide can be encoded by a full-length nucleic acid sequence or any portion of the full-length nucleic acid sequence, so long as a functional activity of the polypeptide is retained, not to include fragments containing only amino acids 1-22 of SEQ ID NO:13 or only amino acids 1-33 of SEQ ID NO:107.
The amino acid sequence will be substantially similar to the sequence shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, or the corresponding full-length amino acid sequence, or fragments thereof, not to include fragments consisting only of the amino acid sequences 1-22 of SEQ ID NO:13 or 1-33 of SEQ ID NO:107. A sequence that is substantially similar to the sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107 will preferably have at least 90% identity (more preferably at least 95% and most preferably 99-100%) to the sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107.
By xe2x80x9cidentityxe2x80x9d is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues by the total number of residues and gaps and multiplying the product by 100. xe2x80x9cGapsxe2x80x9d are spaces in an alignment that are the result of additons or deletions of amino acids. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved, and have deletions, additions, or replacements, may have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity using standard parameters, for example Blast (Altschul, et al. (1997) Nucleic Acids Res. 25:3389-3402), Blast2 (Altschul, et al. (1990) J. mol. biol. 215:403-410), and Smith-Waterman (Smith, et al. (1981) J. Mol. Biol. 147:195-197).
In preferred embodiments, the invention features isolated, enriched, or purified nucleic acid molecules encoding a kinase polypeptide comprising a nucleotide sequence that: (a) encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107; (b) is the complement of the nucleotide sequence of (a); (c) hybridizes under highly stringent conditions to the nucleotide molecule of (a) and encodes a naturally occurring kinase polypeptide; (d) encodes a kinase polypeptide having the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO: 103, SEQ ID NO:105, or SEQ ID NO:107, except that it lacks one or more, but not all, of the following segments of amino acid residues: 1-21, 22-274, or 275-416 of SEQ ID NO:5, 1-31, 32-308, 309-489 or 490-516 of SEQ ID NO:6, 1-178 or 179-414 of SEQ ID NO:7, 1-22, 23-289, 290-526, 527-640, 641-896, or 897-1239 of SEQ ID NO:13, 1-255, 256-442, 443-626, 627-954, or 955-1297 of SEQ ID NO:14, 1-255, 256-476, 477-680, 681-983, or 984-1326 of SEQ ID NO:15, 1-13, 14-273, 274-346, 347-534, or 535-894 of SEQ ID NO:18, 1-21, 22-277, 278-427, 428-637, 638-751, or 752-898 of SEQ ID NO:22, 1-66, 67-215, 216-425, 426-539, 540-786, or 787-887 of SEQ ID NO:23, 1-25, 26-273, 274-422, 423-632, or 633-748 of SEQ ID NO:24, 1-51, 52-224, 225-393, 394-658, or 659-681 of SEQ ID NO:29, 1-25, 26-281, 284-430, 431-640, 641-754, 755-901, or 902-1001 of SEQ ID NO:31, 1-10, 11-321, or 322-373 of SEQ ID NO:97, 1-57, 58-369, or 370-418 of SEQ ID NO:99, 1-52, 53-173, 174-307, 308-572, or 573-591 of SEQ ID NO:103, 1-24, 25-289, 290-397, 398-628, 629-872, or 873-1227 of SEQ ID NO:105, or 1-33, 34-294, 295-337, 338-472, 473-724, or 725-968 of SEQ ID NO:107; (e) is the complement of the nucleotide sequence of (d); (f) encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31; SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107 from amino acid residues 1-21, 22-274, or 275-416 of SEQ ID NO:5, 1-31, 32-308, 309-489, or 490-516 of SEQ ID NO:6, 1-178 or 179-414 of SEQ ID NO:7, 23-289, 290-526, 527-640, 641-896, or 897-1239 of SEQ ID NO:13, 1-255, 256-442, 443-626, 627-954, or 955-1297 of SEQ ID NO:14, 1-255, 256-476, 477-680, 681-983, or 984-1326 of SEQ ID NO:15, 1-13, 14-273, 274-346, 347-534, or 535-894 of SEQ ID NO:18, 1-21, 22-277, 278-427, 428-637, 638-751, or 752-898 of SEQ ID NO:22, 1-66, 67-215, 216-425, 426-539, 540-786, or 787-887 of SEQ ID NO:23, 1-25, 26-273, 274-422, 423-632, or 633-748 of SEQ ID NO:24, 1-51, 52-224, 225-393, 394-658, or 659-681 of SEQ ID NO:29, 1-25, 26-281, 282-430, 431-640, 641-754, 755-901, or 902-1001 of SEQ ID NO:31, 1-10, 11-321, or 322-373 of SEQ ID NO:97, 1-57, 58-369, or 370-418 of SEQ ID NO:99, 1-52, 53-173, 174-307, 308-572, or 573-591 of SEQ ID NO:103, 1-24, 25-289, 290-397, 398-628, 629-872, or 873-1227 of SEQ ID NO:105,or 1-33, 34-294, 295-337, 338-472, 473-724, or 725-968 of SEQ ID NO:107; (g) is the complement of the nucleotide sequence of (f);(h) encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, except that it lacks one or more of the domains selected from the group consisting of a N-terminal domain, a catalytic domain, a C-terminal domain, a coiled-coil structure region, a proline-rich region, a spacer region, an insert, and a C-terminal tail; or (i) is the complement of the nucleotide sequence of (h).
The term xe2x80x9ccomplementxe2x80x9d refers to two nucleotides that can form multiple favorable interactions with one another. For example, adenine is complementary to thymine as they can form two hydrogen bonds. Similarly, guanine and cytosine are complementary since they can form three hydrogen bonds. A nucleotide sequence is the complement of another nucleotide sequence if all of the nucleotides of the first sequence are complementary to all of the nucleotides of the second sequence.
The term xe2x80x9cdomainxe2x80x9d refers to a region of a polypeptide which contains a particular function. For instance, N-terminal or C-terminal domains of signal transduction proteins can serve functions including, but not limited to, binding molecules that localize the signal transduction molecule to different regions of the cell or binding other signaling molecules directly responsible for propagating a particular cellular signal. Some domains can be expressed separately from the rest of the protein and function by themselves, while others must remain part of the intact protein to retain function. The latter are termed functional regions of proteins and also relate to domains.
The term xe2x80x9cN-terminal domainxe2x80x9d refers to the extracatalytic region located between the initiator methionine and the catalytic domain of the protein kinase. The N-terminal domain can be identified following a Smith-Waterman alignment of the protein sequence against the non-redundant protein database to define the N-terminal boundary of the catalytic domain. Depending on its length, the N-terminal domain may or may not play a regulatory role in kinase function. An example of a protein kinase whose N-terminal domain has been shown to play a regulatory role is PAK65, which contains a CRIB motif used for Cdc42 and rac binding (Burbelo, P.D. et al. (1995) J. Biol. Chem. 270, 29071-290740).
The N-terminal domain spans amino acid residues 1-21 of the sequence set forth in SEQ ID NO:5, amino acid residues 1-31 of the sequence set forth in SEQ ID NO:6, amino acid residues 1-22 of the sequence set forth in SEQ ID NO:13, amino acid residues 1-13 of the sequence set forth in SEQ ID NO:18, amino acid residues 1-21 of the sequence set forth in SEQ ID NO:22, amino acid residues 1-25 of the sequence set forth in SEQ ID NO:24, amino acid residues 1-51 of the sequence set forth in SEQ ID NO:29, amino acid residues 1-25 of the sequence set forth in SEQ ID NO:31, amino acid residues 1-57 of the sequence set forth in SEQ ID NO:99, amino acid residues 1-52 of the sequence set forth in SEQ ID NO:103, amino acid residues 1-24 of the sequence set forth in SEQ ID NO:105, or amino acid residues 1-33 of the sequence set forth in SEQ ID NO:107.
The term xe2x80x9ccatalytic domainxe2x80x9d refers to a region of the protein kinase that is typically 25-300 amino acids long and is responsible for carrying out the phosphate transfer reaction from a high-energy phosphate donor molecule such as ATP or GTP to itself (autophosphorylation) or to other proteins (exogenous phosphorylation). The catalytic domain of protein kinases is made up of 12 subdomains that contain highly conserved amino acid residues, and are responsible for proper polypeptide folding and for catalysis. The catalytic domain can be identified following a Smith-Waterman alignment of the protein sequence against the non-redundant protein database.
The catalytic domain spans amino acid residues 22-274 of the sequence set forth in SEQ ID NO:5, residues 32-308 of the sequence set forth in SEQ ID NO:6, residues 1-178 of the sequence set forth in SEQ ID NO:7, residues 23-289 of the sequence set forth in SEQ ID NO:13, residues 1-255 of the sequence set forth in SEQ ID NO:14, residues 1-255 of the sequence set forth in SEQ ID NO:15, residues 14-273 of the sequence set forth in SEQ ID NO:18, residues 22-277 of the sequence set forth in SEQ ID NO:22, residues 1-66 of the sequence set forth in SEQ ID NO:23, residues 26-273 of the sequence set forth in SEQ ID NO:24, residues 394-658 of the sequence set forth in SEQ ID NO:29, residues 26-281 of the sequence set forth in SEQ ID NO:31, residues 1-278 of the sequence set forth in SEQ ID NO:97, residues 58-369 of the sequence set forth in SEQ ID NO:99, residues 1-103 of the sequence set forth in SEQ ID NO:101, residues 308-572 of the sequence set forth in SEQ ID NO:103, residues 25-289 of the sequence set forth in SEQ ID NO:105, or residues 34-294 of the sequence set forth in SEQ ID NO:107.
The term xe2x80x9ccatalytic activityxe2x80x9d, as used herein, defines the rate at which a kinase catalytic domain phosphorylates a substrate. Catalytic activity can be measured, for example, by determining the amount of a substrate converted to a phosphorylated product as a function of time. Catalytic activity can be measured by methods of the invention by holding time constant and determining the concentration of a phosphorylated substrate after a fixed period of time. Phosphorylation of a substrate occurs at the active-site of a protein kinase. The active-site is normally a cavity in which the substrate binds to the protein kinase and is phosphorylated.
The term xe2x80x9csubstratexe2x80x9d as used herein refers to a molecule phosphorylated by a kinase of the invention. Kinases phosphorylate substrates on serine/threonine or tyrosine amino acids. The molecule may be another protein or a polypeptide.
The term xe2x80x9cC-terminal domainxe2x80x9d refers to the region located between the catalytic domain or the last (located closest to the C-terminus) functional domain and the carboxy-terminal amino acid residue of the protein kinase. By xe2x80x9cfunctionalxe2x80x9d domain is meant any region of the polypeptide that may play a regulatory or catalytic role as predicted from amino acid sequence homology to other proteins or by the presence of amino acid sequences that may give rise to specific structural conformations (i.e. coiled-coils). The C-terminal domain can be identified by using a Smith-Waterman alignment of the protein sequence against the non-redundant protein database to define the C-terminal boundary of the catalytic domain or of any functional C-terminal extracatalytic domain. Depending on its length and amino acid composition, the C-terminal domain may or may not play a regulatory role in kinase function. An example of a protein kinase whose C-terminal domain may play a regulatory role is PAK3 which contains a heterotrimeric Gb subunit-binding site near its C-terminus (Leeuw, T. et al (1998) Nature, 391, 191-195).
The C-terminal domain spans amino acid residues 275-416 of the sequence set forth in SEQ ID NO:5, residues 309-489 of the sequence set forth in SEQ ID NO:6, residues 179-414 of the sequence set forth in SEQ ID NO:7, residues 897-1239 of the sequence set forth in SEQ ID NO:13, residues 955-1297 of the sequence set forth in SEQ ID NO:14, residues 984-1326 of the sequence set forth in SEQ ID NO:15, residues 535-894 of the sequence set forth in SEQ ID NO:18, residues 752-898 of the sequence set forth in SEQ ID NO:22, residues 279-330 of the sequence set forth in SEQ ID NO:97, residues 370-418 of the sequence set forth in SEQ ID NO:99, or residues 873-1227 of the sequence set forth in SEQ ID NO:105.
The term xe2x80x9csignal transduction pathwayxe2x80x9d refers to the molecules that propagate an extracellular signal through the cell membrane to become an intracellular signal. This signal can then stimulate a cellular response. The polypeptide molecules involved in signal transduction processes are typically receptor and non-receptor protein tyrosine kinases, receptor and non-receptor protein phosphatases, SRC homology 2 and 3 domains, phosphotyrosine binding proteins (SRC homology 2 (SH2) and phosphotyrosine binding (PTB and PH) domain containing proteins), proline-rich binding proteins (SH3 domain containing proteins), nucleotide exchange factors, and transcription factors.
The term xe2x80x9ccoiled-coil structure regionxe2x80x9d as used herein, refers to a polypeptide sequence that has a high probability of adopting a coiled-coil structure as predicted by computer algorithms such as COILS (Lupas, A. (1996) Meth. Enzymology 266:513-525). Coiled-coils are formed by two or three amphipathic xcex1-helices in parallel. Coiled-coils can bind to coiled-coil domains of other polypeptides resulting in homo- or heterodimers (Lupas, A. (1991) Science 252:1162-1164). Coiled-coil-dependent oligomerization has been shown to be necessary for protein function including catalytic activity of serine/threonine kinases (Roe, J. et al. (1997) J. Biol. Chem. 272:5838-5845).
The coiled-coil structure region spans amino acid residues 290-526 of the sequence set forth in SEQ ID NO:13, residues 256-442 of the sequence set forth in SEQ ID NO:14, residues 256-476 of the sequence set forth in SEQ ID NO:15, residues 428-637 of the sequence set forth in SEQ ID NO:22, residues 216-425 or 540-786 of the sequence set forth in SEQ ID NO:23, residues 423-632 of the sequence set forth in SEQ ID NO:24, residues 431-640 or 755-901 of the sequence set forth in SEQ ID NO:31, residues 291-398 or 629-668 of the sequence set forth in SEQ ID NO:105, or residues 473-724 or 725-968 of the sequence set forth in SEQ ID NO:107.
The term xe2x80x9cproline-rich regionxe2x80x9d as used herein, refers to a region of a protein kinase whose proline content over a given amino acid length is higher than the average content of this amino acid found in proteins (i.e.,  greater than 10%). Proline-rich regions are easily discernable by visual inspection of amino acid sequences and quantitated by standard computer sequence analysis programs such as the DNAStar program EditSeq. Proline-rich regions have been demonstrated to participate in regulatory proteinxe2x80x94protein interactions. Among these interactions, those that are most relevant to this invention involve the xe2x80x9cPxxPxe2x80x9d (SEQ ID NO: 148) proline rich motif found in certain protein kinases (i.e., human PAM) and the SH3 domain of the adaptor molecule Nck (Galisteo, M. L. et al. (1996) J. Biol. Chem. 271:20997-21000). Other regulatory interactions involving xe2x80x9cPxxPxe2x80x9d (SEQ ID NO: 148) proline-rich motifs include the WW domain (Sudol, M. (1996) Prog. Biochys. Mol. Bio. 65:113-132).
The proline-rich region spans amino acid residues 527-640 of the sequence set forth in SEQ ID NO:13, residues 443-626 of the sequence set forth in SEQ ID NO:14, residues 477-680 of the sequence set forth in SEQ ID NO:15, residues 347-534 of the sequence set forth in SEQ ID NO:18,residues 398-628 of the sequence set forth in SEQ ID NO:105, or residues 338-472 of the sequence set forth in SEQ ID NO:107.
The term xe2x80x9cspacer regionxe2x80x9d as used herein, refers to a region of the protein kinase located between predicted functional domains. The spacer region has no detectable homology to any amino acid sequence in the database, and can be identified by using a Smith-Waterman alignment of the protein sequence against the non-redundant protein database to define the C- and N-terminal boundaries of the flanking functional domains. Spacer regions may or may not play a fundamental role in protein kinase function. Precedence for the regulatory role of spacer regions in kinase function is provided by the role of the src kinase spacer in inter-domain interactions (Xu, W. et al. (1997) Nature 385:595-602).
The spacer region spans amino acid residues 641-896 of the sequence set forth in SEQ ID NO:13, residues 627-954 of the sequence set forth in SEQ ID NO:14, residues 681-983 of the sequence set forth in SEQ ID NO:15, residues 274-346 of the sequence set forth in SEQ ID NO:18, residues 278-427 or 638-751 of the sequence set forth in SEQ ID NO:22, residues 67-215 or 426-539 of the sequence set forth in SEQ ID NO:23, residues 274-422 or 633-748 of the sequence set forth in SEQ ID NO:24, residues 225-393 of the sequence set forth in SEQ ID NO:29, residues 282-430 or 641-754 of the sequence set forth in SEQ ID NO:31, residues 174-307 of the sequence set forth in SEQ ID NO:103, residues 669-872 of the sequence set forth in SEQ ID NO:105, or residues 295-337 of the sequence set forth in SEQ ID NO:107.
The term xe2x80x9cinsertxe2x80x9d as used herein refers to a portion of a protein kinase that is absent from a close homolog. Inserts may or may not by the product alternative splicing of exons. Inserts can be identified by using a Smith-Waterman sequence alignment of the protein sequence against the non-redundant protein database, or by means of a multiple sequence alignment of homologous sequences using the DNAStar program Megalign. Inserts may play a functional role by presenting a new interface for proteinxe2x80x94protein interactions, or by interfering with such interactions. Inserts span amino acid residues 52-224 of the sequence set forth in SEQ ID NO:29 or residues 53-173 of the sequence set forth in SEQ ID NO:103.
The term xe2x80x9cC-terminal tailxe2x80x9d as used herein, refers to a C-terminal domain of a protein kinase, that by homology extends or protrudes past the C-terminal amino acid of its closest homolog. C-terminal tails can be identified by using a Smith-Waterman sequence alignment of the protein sequence against the non-redundant protein database, or by means of a multiple sequence alignment of homologous sequences using the DNAStar program Megalign. Depending on its length, a C-terminal tail may or may not play a regulatory role in kinase function.
The C-terminal tail spans amino acid residues 490-516 of the sequence set forth in SEQ ID NO:6, residues 787-887 of the sequence set forth in SEQ ID NO:23, residues 659-681 of the sequence set forth in SEQ ID NO:29, residues 994-1093 of the sequence set forth in SEQ ID NO:31, or residues 573-591 of the sequence set forth in SEQ ID NO:103.
Various low or high stringency hybridization conditions may be used depending upon the specificity and selectivity desired. These conditions are well-known to those skilled in the art. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 20 contiguous nucleotides, more preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 50 contiguous nucleotides, most preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 100 contiguous nucleotides. In some instances, the conditions may prevent hybridization of nucleic acids having more than 5 mismatches in the full-length sequence.
By stringent hybridization assay conditions is meant hybridization assay conditions at least as stringent as the following: hybridization in 50% formamide, 5xc3x97 SSC, 50 mM NaH2PO4, pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5xc3x97 Denhart solution at 42xc2x0 C. overnight; washing with 2xc3x97 SSC, 0.1% SDS at 45xc2x0 C.; and washing with 0.2xc3x97 SSC, 0.1% SDS at 45xc2x0 C. Under some of the most stringent hybridization assay conditions, the second wash can be done with 0.1xc3x97 SSC at a temperature up to 70xc2x0 C. (Berger et al. (1987) Guide to Molecular Cloning Techniques pg 421, hereby incorporated by reference herein including any figures, tables, or drawings.). However, other applications may require the use of conditions falling between these sets of conditions. Methods of determining the conditions required to achieve desired hybridizations are well-known to those with ordinary skill in the art, and are based on several factors, including but not limited to, the sequences to be hybridized and the samples to be tested.
In other preferred embodiments, the invention features isolated, enriched, or purified nucleic acid molecules encoding kinase polypeptides, further comprising a vector or promoter effective to initiate transcription in a host cell. The invention also features recombinant nucleic acid, preferably in a cell or an organism. The recombinant nucleic acid may contain a sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102,SEQ ID NO:104, or SEQ ID NO:106, or a functional derivative thereof and a vector or a promoter effective to initiate transcription in a host cell. The recombinant nucleic acid can alternatively contain a transcriptional initiation region functional in a cell, a sequence complementary to an RNA sequence encoding a kinase polypeptide and a transcriptional termination region functional in a cell. Specific vectors and host cell combinations are discussed herein.
The term xe2x80x9cvectorxe2x80x9d relates to a single or double-stranded circular nucleic acid molecule that can be transfected into cells and replicated within or independently of a cell genome. A circular double-stranded nucleic acid molecule can be cut and thereby linearized upon treatment with restriction enzymes. An assortment of nucleic acid vectors, restriction enzymes, and the knowledge of the nucleotide sequences cut by restriction enzymes are readily available to those skilled in the art. A nucleic acid molecule encoding a kinase can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together.
The term xe2x80x9ctransfectingxe2x80x9d defines a number of methods to insert a nucleic acid vector or other nucleic acid molecules into a cellular organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt, an electric field, detergent, or DMSO to render the outer membrane or wall of the cells permeable to nucleic acid molecules of interest or use of various viral transduction strategies.
The term xe2x80x9cpromoterxe2x80x9d as used herein, refers to nucleic acid sequence needed for gene sequence expression. Promoter regions vary from organism to organism, but are well known to persons skilled in the art for different organisms. For example, in prokaryotes, the promoter region contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal synthesis initiation. Such regions will normally include those 5xe2x80x2-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
In preferred embodiments, the isolated nucleic acid comprises, consists essentially of, or consists of a nucleic acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100 SEQ ID NO:102, SEQ ID NO:104, or SEQ ID NO:106, or the corresponding full-length sequence, encodes the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, or the corresponding full-length amino acid sequence, a functional derivative thereof, or at least 40, 45, 50, 60, 100, 200, or 300 contiguous amino acids of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or of the corresponding full-length amino acid sequence; at least 250, 255, 275, 300, or 400 contiguous amino acids of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or of the corresponding full-length amino acid sequence; at least 27, 30, 35, 40, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:18; at least 16, 25, 35, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:103, or of the corresponding full-length amino acid sequence; 6 (preferably 10, more preferably 15, most preferably 25) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:97 or SEQ ID NO:99, or the corresponding full-length amino acid sequence; 22 (preferably 30, more preferably 35, most preferably 45) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:101, or the corresponding full-length amino acid sequence; or at least 80, 85, 90, 100, 200, or 300 contiguous amino acids of SEQ ID NO:107, or functional derivatives thereof. The kinase polypeptides, selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5, comprise, consist essentially of, or consist of at least at least 40, 45, 50, 60, 100, 200, or 300 contiguous amino acids of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; at least 250, 255, 275, 300, or 400 contiguous amino acids of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:105; at least 27, 30, 35, 40, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:18; at least 35, 40, 45, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:103; 6 (preferably 10, more preferably 15, most preferably 25) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:97 or SEQ ID NO:99; 22 (preferably 30, more preferably 35, most preferably 45) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:101; or at least 80, 85, 90, 100, 200, or 300 contiguous amino acids of SEQ ID NO:107, or the corresponding full-length sequences or derivatives thereof. The nucleic acid may be isolated from a natural source by cDNA cloning or by subtractive hybridization. The natural source may be mammalian, preferably human, blood, semen, or tissue, and the nucleic acid may be synthesized by the triester method or by using an automated DNA synthesizer.
The term xe2x80x9cmammalxe2x80x9d refers preferably to such organisms as mice, rats, rabbits, guinea pigs, sheep, and goats, more preferably to cats, dogs, monkeys, and apes, and most preferably to humans.
In yet other preferred embodiments, the nucleic acid is a conserved or unique region, for example those useful for: the design of hybridization probes to facilitate identification and cloning of additional polypeptides, the design of PCR probes to facilitate cloning of additional polypeptides, obtaining antibodies to polypeptide regions, and designing antisense oligonucleotides.
By xe2x80x9cconserved nucleic acid regionsxe2x80x9d, are meant regions present on two or more nucleic acids encoding a kinase polypeptide, to which a particular nucleic acid sequence can hybridize under lower stringency conditions. Examples of lower stringency conditions suitable for screening for nucleic acid encoding kinase polypeptides are provided in Abe, et al. (J. Biol. Chem. 19:13361-13368, 1992), hereby incorporated by reference herein in its entirety, including any drawings, figures, or tables. Preferably, conserved regions differ by no more than 5 out of 20 nucleotides, even more preferably 2 out of 20 nucleotides or most preferably 1 out of 20 nucleotides.
By xe2x80x9cunique nucleic acid regionxe2x80x9d is meant a sequence present in a nucleic acid coding for a kinase polypeptide that is not present in a sequence coding for any other naturally occurring polypeptide. Such regions preferably encode 32 (preferably 40, more preferably 45, most preferably 55) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or the corresponding full-length amino acid sequence; 250 (preferably 255, more preferably 260, most preferably 270) or more contiguous amino acids set forth in the amino acid sequence SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15, or SEQ ID NO:105, or the corresponding full-length amino acid sequence; 27 (preferably 30, more preferably 40, most preferably 45) or more contiguous amino acids set forth in the amino acid sequence SEQ ID NO:18; 16 (preferably 20, more preferably 25, most preferably 35) or more contiguous amino acids set forth in the amino acid sequence SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:103, or the corresponding full-length amino acid sequence; 6 (preferably 10, more preferably 15, most preferably 25) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:97 or SEQ ID NO:99, 22 (preferably 30, more preferably 35, most preferably 45) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:101, or the corresponding full-length amino acid sequence; or 78 (preferably 80, more preferably 85, most preferably 90) or more contiguous amino acids set forth in the amino acid sequence SEQ ID NO:107, or functional derivatives thereof. In particular, a unique nucleic acid region is preferably of mammalian origin.
A second aspect of the invention features a nucleic acid probe for the detection of nucleic acid encoding a kinase polypeptide in a sample, wherein said polypeptide is selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5. Preferably, the nucleic acid probe encodes a kinase polypeptide that is a fragment of the protein encoded by the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, or the corresponding full-length amino acid sequences, not to include fragments consisting only of amino acids 1-22 of SEQ ID NO:13 or amino acids 1-33 of SEQ ID NO:107. The nucleic acid probe contains a nucleotide base sequence that will hybridize to a sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, or SEQ ID NO:106, or the corresponding full-length sequence, or a functional derivative thereof.
In preferred embodiments, the nucleic acid probe hybridizes to nucleic acid encoding at least 6, 12, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids of the sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31 SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, or the corresponding full-length amino acid sequence, or functional derivatives thereof.
Methods for using the probes include detecting the presence or amount of kinase RNA in a sample by contacting the sample with a nucleic acid probe under conditions such that hybridization occurs and detecting the presence or amount of the probe bound to kinase RNA. The nucleic acid duplex formed between the probe and a nucleic acid sequence coding for a kinase polypeptide may be used in the identification of the sequence of the nucleic acid detected (Nelson et al., in Nonisotopic DNA Probe Techniques, Academic Press, San Diego, Kricka, ed., p. 275, 1992, hereby incorporated by reference herein in its entirety, including any drawings, figures, or tables). Kits for performing such methods may be constructed to include a container means having disposed therein a nucleic acid probe.
In a third aspect, the invention describes a recombinant cell or tissue comprising a nucleic acid molecule encoding a kinase polypeptide selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5. In such cells, the nucleic acid may be under the control of the genomic regulatory elements, or may be under the control of exogenous regulatory elements including an exogenous promoter. By xe2x80x9cexogenousxe2x80x9d it is meant a promoter that is not normally coupled in vivo transcriptionally to the coding sequence for the kinase polypeptides.
The polypeptide is preferably a fragment of the protein encoded by the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, or the corresponding full-length amino acid sequence, not to include fragments consisting only of amino acids 1-22 of SEQ ID NO:13 or amino acids 1-33 of SEQ ID NO:107. By xe2x80x9cfragment,xe2x80x9d is meant an amino acid sequence present in a kinase polypeptide. Preferably, such a sequence comprises at least 32, 45, 50, 60, 100, 200, or 300 contiguous amino acids of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or of the corresponding full-length amino acid sequence; at least 250, 255, 275, 300, or 400 contiguous amino acids of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, OR SEQ ID NO:105, or of the corresponding full-length amino acid sequence; at least 27, 30, 35, 40, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:18; at least 16, 25, 35, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:103, or of the corresponding full-length amino acid sequence; 6 (preferably 10, more preferably 15, most preferably 25) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:97 or SEQ ID NO:99, 22 (preferably 30, more preferably 35, most preferably 45) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:101; at least 78, 85, 90, 100, 200, or 300 contiguous amino acids of SEQ ID NO:107, or the corresponding full-length amino acid sequence; or a functional derivative thereof.
In a fourth aspect, the invention features an isolated, enriched, or purified kinase polypeptide selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5.
By xe2x80x9cisolatedxe2x80x9d in reference to a polypeptide is meant a polymer of amino acids (2 or more amino acids) conjugated to each other, including polypeptides that are isolated from a natural source or that are synthesized. The isolated polypeptides of the present invention are unique in the sense that they are not found in a pure or separated state in nature. Use of the term xe2x80x9cisolatedxe2x80x9d indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only amino acid chain present, but that it is essentially free (about 90-95% pure at least) of non-amino acid material naturally associated with it.
By the use of the term xe2x80x9cenrichedxe2x80x9d in reference to a polypeptide is meant that the specific amino acid sequence constitutes a significantly higher fraction (2-5 fold) of the total amino acid sequences present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other amino acid sequences present, or by a preferential increase in the amount of the specific amino acid sequence of interest, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other amino acid sequences present, just that the relative amount of the sequence of interest has been significantly increased. The term significant here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other amino acid sequences of about at least 2-fold, more preferably at least 5- to 10-fold or even more. The term also does not imply that there is no amino acid sequence from other sources. The other source of amino acid sequences may, for example, comprise amino acid sequence encoded by a yeast or bacterial genome, or a cloning vector such as pUC19. The term is meant to cover only those situations in which man has intervened to increase the proportion of the desired amino acid sequence.
It is also advantageous for some purposes that an amino acid sequence be in purified form. The term xe2x80x9cpurifiedxe2x80x9d in reference to a polypeptide does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment. Compared to the natural level this level should be at least 2-5 fold greater (e.g., in terms of mg/mL). Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. The substance is preferably free of contamination at a functionally significant level, for example 90%, 95%, or 99% pure.
In preferred embodiments, the kinase polypeptide is a fragment of the protein encoded by the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, or the corresponding full-length amino acid sequences, not to include fragments consisting only of amino acids 1-22 of SEQ ID NO:13 or amino acids 1-33 of SEQ ID NO:107. Preferably, the kinase polypeptide contains at least 32, 45, 50, 60, 100, 200, or 300 contiguous amino acids of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or the corresponding full-length amino acid sequence; at least 250, 255, 275, 300, or 400 contiguous amino acids of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:105, or the corresponding full-length amino acid sequence; at least 27, 30, 35, 40, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:18; at least 16, 25, 35, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:103, or the corresponding full-length amino acid sequence; 6 (preferably 10, more preferably 15, most preferably 25) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:97 or SEQ ID NO:99, 22 (preferably 30, more preferably 35, most preferably 45) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:101, or the corresponding full-length amino acid sequence; or at least 78, 85, 90, 100, 200, or 300 contiguous amino acids of SEQ ID NO:107, or a functional derivative thereof.
In preferred embodiments, the kinase polypeptide comprises an amino acid sequence having (a) the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107; (b) the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, except that it lacks one or more, but not all, of the following segments of amino acid residues: 1-21, 22-274, or 275-416 of SEQ ID NO:5, 1-31, 32-308, 309-489 or 490-516 of SEQ ID NO:6, 1-178 or 179-414 of SEQ ID NO:7, 1-22, 23-289, 290-526, 527-640, 641-896, or 897-1239 of SEQ ID NO:13, 1-255, 256-442, 443-626, 627-954, or 955-1297 of SEQ ID NO:14, 1-255, 256-476, 477-680, 681-983, or 984-1326 of SEQ ID NO:15, 1-13, 14-273, 274-346, 347-534, or 535-894 of SEQ ID NO:18, 1-21, 22-277, 278-427, 428-637, 638-751, or 752-898 of SEQ ID NO:22, 1-66, 67-215, 216-425, 426-539, 540-786, or 787-887 of SEQ ID NO:23, 1-25, 26-273, 274-422, 423-632, or 633-748 of SEQ ID NO:24, 1-51, 52-224, 225-393, 394-658, or 659-681 of SEQ ID NO:29, 1-25, 26-281, 282-430, 431-640, 641-754, 755-901, or 902-1001 of SEQ ID NO:31, 1-10, 11-321, or 322-373 of SEQ ID NO:97, 1-57, 58-369, or 370-418 of SEQ ID NO:99, 1-52, 53-173, 174-307, 308-572, or 573-591 of SEQ ID NO:103, 1-24, 25-289, 290-397, 398-628, 629-668, 669-872, or 873-1227 of SEQ ID NO:105, or 1-33, 34-294, 295-337, 338-472, 473-724, or 725-968 of SEQ ID NO:107; (c) the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107 from amino acid residues 1-21, 22-274, or 275-416 of SEQ ID NO:5, 1-31, 32-308, 309-489, or 490-516 of SEQ ID NO:6, 1-178 or 179-414 of SEQ ID NO:7, 23-289, 290-526, 527-640, 641-896, or 897-1239 of SEQ ID NO:13, 1-255, 256-442, 443-626, 627-954, or 955-1297 of SEQ ID NO:14, 1-255, 256-476, 477-680, 681-983, or 984-1326 of SEQ ID NO:15, 1-13, 14-273, 274-346, 347-534, or 535-894 of SEQ ID NO:18, 1-21, 22-277, 278-427, 428-637, 638-751, or 752-898 of SEQ ID NO:22, 1-66, 67-215, 216-425, 426-539, 540-786, or 787-887 of SEQ ID NO:23, 1-25, 26-273, 274-422, 423-632, or 633-748 of SEQ ID NO:24, 1-51, 52-224, 225-393, 394-658, or 659-681 of SEQ ID NO:29, 1-25, 26-273, 274-422, 423-632, 633-746, 747-993, or 994-1093 of SEQ ID NO:31, 1-10, 11-321, or 322-373 of SEQ ID NO:97, 1-57, 58-369, or 370-418 of SEQ ID NO:99, 1-52, 53-173, 174-307, 308-572, or 573-591 of SEQ ID NO:103, 1-24, 25-289, 290-397, 398-628, 629-668, 669-872, or 873-1227 of SEQ ID NO:105, or 1-33, 34-294, 295-337, 338-472, 473-724, or 725-968 of SEQ ID NO:107; or (d) the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, except that it lacks one or more, but not all, of the domains selected from the group consisting of a C-terminal domain, a catalytic domain, an N-terminal domain, a spacer region, a proline-rich region, a coiled-coil structure region, an insert, and a C-terminal tail.
The polypeptide can be isolated from a natural source by methods well-known in the art. The natural source may be mammalian, preferably human, blood, semen, or tissue, and the polypeptide may be synthesized using an automated polypeptide synthesizer. The isolated, enriched, or purified kinase polypeptide is preferably: a STLK2, STLK3, STLK4, STLK5, STLK6, or STLK7 polypeptide; a ZC1, ZC2, ZC3, or ZC4 polypeptide; a KHS2 polypeptide; a SULU1 or SULU3 polypeptide; a GEK2 polypeptide; or a PAK4 or PAK5 polypeptide.
In some embodiments the invention includes a recombinant kinase polypeptide selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5. By xe2x80x9crecombinant kinase polypeptidexe2x80x9d is meant a polypeptide produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location (e.g., present in a different cell or tissue than found in nature), purity or structure. Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature.
In a fifth aspect, the invention features an antibody (e.g., a monoclonal or polyclonal antibody) having specific binding affinity to a kinase polypeptide or a kinase polypeptide domain or fragment where the polypeptide is selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5. By xe2x80x9cspecific binding affinityxe2x80x9d is meant that the antibody binds to the target kinase polypeptide with greater affinity than it binds to other polypeptides under specified conditions. Antibodies or antibody fragments are polypeptides that contain regions that can bind other polypeptides. The term xe2x80x9cspecific binding affinityxe2x80x9d describes an antibody that binds to a kinase polypeptide with greater affinity than it binds to other polypeptides under specified conditions.
The term xe2x80x9cpolyclonalxe2x80x9d refers to antibodies that are heterogenous populations of antibody molecules derived from the sera of animals immunized with an antigen or an antigenic functional derivative thereof. For the production of polyclonal antibodies, various host animals may be immunized by injection with the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species.
xe2x80x9cMonoclonal antibodiesxe2x80x9d are substantially homogenous populations of antibodies to a particular antigen. They may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. Monoclonal antibodies may be obtained by methods known to those skilled in the art (Kohler et al., Nature 256:495-497, 1975, and U.S. Pat. No. 4,376,110, both of which are hereby incorporated by reference herein in their entirety including any figures, tables, or drawings).
The term xe2x80x9cantibody fragmentxe2x80x9d refers to a portion of an antibody, often the hyper variable region and portions of the surrounding heavy and light chains, that displays specific binding affinity for a particular molecule. A hyper variable region is a portion of an antibody that physically binds to the polypeptide target.
Antibodies or antibody fragments having specific binding affinity to a kinase polypeptide of the invention may be used in methods for detecting the presence and/or amount of kinase polypeptide in a sample by probing the sample with the antibody under conditions suitable for kinase-antibody immunocomplex formation and detecting the presence and/or amount of the antibody conjugated to the kinase polypeptide. Diagnostic kits for performing such methods may be constructed to include antibodies or antibody fragments specific for the kinase as well as a conjugate of a binding partner of the antibodies or the antibodies themselves.
An antibody or antibody fragment with specific binding affinity to a kinase polypeptide of the invention can be isolated, enriched, or purified from a prokaryotic or eukaryotic organism. Routine methods known to those skilled in the art enable production of antibodies or antibody fragments, in both prokaryotic and eukaryotic organisms. Purification, enrichment, and isolation of antibodies, which are polypeptide molecules, are described above.
Antibodies having specific binding affinity to a kinase polypeptide of the invention may be used in methods for detecting the presence and/or amount of kinase polypeptide in a sample by contacting the sample with the antibody under conditions such that an immunocomplex forms and detecting the presence and/or amount of the antibody conjugated to the kinase polypeptide. Diagnostic kits for performing such methods may be constructed to include a first container containing the antibody and a second container having a conjugate of a binding partner of the antibody and a label, such as, for example, a radioisotope. The diagnostic kit may also include notification of an FDA approved use and instructions therefor.
In a sixth aspect, the invention features a hybridoma which produces an antibody having specific binding affinity to a kinase polypeptide or a kinase polypeptide domain, where the polypeptide is selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5. By xe2x80x9chybridomaxe2x80x9d is meant an immortalized cell line that is capable of secreting an antibody, for example an antibody to a kinase of the invention. In preferred embodiments, the antibody to the kinase comprises a sequence of amino acids that is able to specifically bind a kinase polypeptide of the invention.
In a seventh aspect, the invention features a kinase polypeptide binding agent able to bind to a kinase polypeptide selected from the group consisting of STLK2, STLK3, STLK4, STLK6, STLK7, STLK5, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5. The binding agent is preferably a purified antibody that recognizes an epitope present on a kinase polypeptide of the invention. Other binding agents include molecules that bind to kinase polypeptides and analogous molecules that bind to a kinase polypeptide. Such binding agents may be identified by using assays that measure kinase binding partner activity, such as those that measure PDGFR activity.
The invention also features a method for screening for human cells containing a kinase polypeptide of the invention or an equivalent sequence. The method involves identifying the novel polypeptide in human cells using techniques that are routine and standard in the art, such as those described herein for identifying the kinases of the invention (e.g., cloning, Southern or Northern blot analysis, in situ hybridization, PCR amplification, etc.).
In an eighth aspect, the invention features methods for identifying a substance that modulates kinase activity comprising the steps of: (a) contacting a kinase polypeptide selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5 with a test substance; (b) measuring the activity of said polypeptide; and (c) determining whether said substance modulates the activity of said polypeptide.
The term xe2x80x9cmodulatesxe2x80x9d refers to the ability of a compound to alter the function of a kinase of the invention. A modulator preferably activates or inhibits the activity of a kinase of the invention depending on the concentration of the compound exposed to the kinase.
The term xe2x80x9cactivatesxe2x80x9d refers to increasing the cellular activity of the kinase. The term inhibit refers to decreasing the cellular activity of the kinase. Kinase activity is preferably the interaction with a natural binding partner.
The term xe2x80x9cmodulatesxe2x80x9d also refers to altering the function of kinases of the invention by increasing or decreasing the probability that a complex forms between the kinase and a natural binding partner. A modulator preferably increases the probability that such a complex forms between the kinase and the natural binding partner, more preferably increases or decreases the probability that a complex forms between the kinase and the natural binding partner depending on the concentration of the compound exposed to the kinase, and most preferably decreases the probability that a complex forms between the kinase and the natural binding partner.
The term xe2x80x9ccomplexxe2x80x9d refers to an assembly of at least two molecules bound to one another. Signal transduction complexes often contain at least two protein molecules bound to one another. For instance, a protein tyrosine receptor protein kinase, GRB2, SOS, RAF, and RAS assemble to form a signal transduction complex in response to a mitogenic ligand.
The term xe2x80x9cnatural binding partnerxe2x80x9d refers to polypeptides, lipids, small molecules, or nucleic acids that bind to kinases in cells. A change in the interaction between a kinase and a natural binding partner can manifest itself as an increased or decreased probability that the interaction forms, or an increased or decreased concentration of kinase/natural binding partner complex.
The term xe2x80x9ccontactingxe2x80x9d as used herein refers to mixing a solution comprising the test compound with a liquid medium bathing the cells of the methods. The solution comprising the compound may also comprise another component, such as dimethyl sulfoxide (DMSO), which facilitates the uptake of the test compound or compounds into the cells of the methods. The solution comprising the test compound may be added to the medium bathing the cells by utilizing a delivery apparatus, such as a pipet-based device or syringe-based device.
In a ninth aspect, the invention features methods for identifying a substance that modulates kinase activity in a cell comprising the steps of: (a) expressing a kinase polypeptide in a cell, wherein said polypeptide is selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5; (b) adding a test substance to said cell; and (c) monitoring a change in cell phenotype or the interaction between said polypeptide and a natural binding partner.
The term xe2x80x9cexpressingxe2x80x9d as used herein refers to the production of kinases of the invention from a nucleic acid vector containing kinase genes within a cell. The nucleic acid vector is transfected into cells using well known techniques in the art as described herein.
In a tenth aspect, the invention provides methods for treating a disease by administering to a patient in need of such treatment a substance that modulates the activity of a kinase selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5. Preferably, the disease is selected from the group consisting of immune-related diseases and disorders, organ transplantation, myocardial infarction, cardiovascular disease, stroke, renal failure, oxidative stress-related neurodegenerative disorders, and cancer. Most preferably, the immune-related diseases and disorders include, but are not limited to, rheumatoid arthritis, artherosclerosis, and autoimmune disorders.
In preferred embodiments, the invention provides methods for treating or preventing a disease or disorder by administering to a patient in need of such treatment a substance that modulates the activity of a kinase polypeptide selected from the group consisting of ZC1, ZC2, ZC3, ZC4, KHS2, PAK4, and PAK5. Preferably, the disease or disorder is selected from the group consisting of rheumatoid arthritis, artherosclerosis, autoimmune disorders, and organ transplantation. The invention also features methods of treating or preventing a disease or disorder by administering to a patient in need of such treatment a substance that modulates the activity of a kinase polypeptide selected from the group consisting of STLK1, STLK2, STLK3, STLK4, STLK5, STLK6, and STLK7. Preferably the disease or disorder is selected from the group consisting of immune-related diseases and disorders, myocardial infarction, cardiomyopathies, stroke, renal failure, and oxidative stress-related neurodegenerative disorders. Most preferably, the immune-related diseases and disorders are selected from the group consisting of rheumatoid arthritis, chronic inflammatory bowel disease, chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis, autoimmunity, and organ transplantation.
The invention also features methods of treating or preventing a disease or disorder by administering to a patient in need of such treatment a substance that modulates the activity of a kinase polypeptide selected from the group consisting of ZC1, ZC2, ZC3, and ZC4. Preferably the disease is selected from the group consisting of immune-related diseases and disorders, cardiovascular disease, and cancer. Most preferably, the immune-related diseases and disorders are selected from the group consisting of rheumatoid arthritis, chronic inflammatory bowel disease, chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis, autoimmunity, and organ transplantation.
Substances useful for treatment of kinase-related disorders or diseases preferably show positive results in one or more in vitro assays for an activity corresponding to treatment of the disease or disorder in question (Examples of such assays are provided in the references in section VI, below; and in Example 7, herein). Examples of substances that can be screened for favorable activity are provided and referenced in section VI, below. The substances that modulate the activity of the kinases preferably include, but are not limited to, antisense oligonucleotides and inhibitors of protein kinases, as determined by methods and screens referenced in section VI and Example 7, below.
The term xe2x80x9cpreventingxe2x80x9d refers to decreasing the probability that an organism contracts or develops an abnormal condition.
The term xe2x80x9ctreatingxe2x80x9d refers to having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism.
The term xe2x80x9ctherapeutic effectxe2x80x9d refers to the inhibition or activation factors causing or contributing to the abnormal condition. A therapeutic effect relieves to some extent one or more of the symptoms of the abnormal condition. In reference to the treatment of abnormal conditions, a therapeutic effect can refer to one or more of the following: (a) an increase in the proliferation, growth, and/or differentiation of cells; (b) inhibition (i.e., slowing or stopping) of cell death; (c) inhibition of degeneration; (d) relieving to some extent one or more of the symptoms associated with the abnormal condition; and (e) enhancing the function of the affected population of cells. Compounds demonstrating efficacy against abnormal conditions can be identified as described herein.
The term xe2x80x9cabnormal conditionxe2x80x9d refers to a function in the cells or tissues of an organism that deviates from their normal functions in that organism. An abnormal condition can relate to cell proliferation, cell differentiation, or cell survival.
Abnormal cell proliferative conditions include cancers such as fibrotic and mesangial disorders, abnormal angiogenesis and vasculogenesis, wound healing, psoriasis, diabetes mellitus, and inflammation.
Abnormal differentiation conditions include, but are not limited to neurodegenerative disorders, slow wound healing rates, and slow tissue grafting healing rates.
Abnormal cell survival conditions relate to conditions in which programmed cell death (apoptosis) pathways are activated or abrogated. A number of protein kinases are associated with the apoptosis pathways. Aberrations in the function of any one of the protein kinases could lead to cell immortality or premature cell death.
The term xe2x80x9caberrationxe2x80x9d, in conjunction with the function of a kinase in a signal transduction process, refers to a kinase that is over- or under-expressed in an organism, mutated such that its catalytic activity is lower or higher than wild-type protein kinase activity, mutated such that it can no longer interact with a natural binding partner, is no longer modified by another protein kinase or protein phosphatase, or no longer interacts with a natural binding partner.
The term xe2x80x9cadministeringxe2x80x9d relates to a method of incorporating a compound into cells or tissues of an organism. The abnormal condition can be prevented or treated when the cells or tissues of the organism exist within the organism or outside of the organism. Cells existing outside the organism can be maintained or grown in cell culture dishes. For cells harbored within the organism, many techniques exist in the art to administer compounds, including (but not limited to) oral, parenteral, dermal, injection, and aerosol applications. For cells outside of the organism, multiple techniques exist in the art to administer the compounds, including (but not limited to) cell microinjection techniques, transformation techniques, and carrier techniques.
The abnormal condition can also be prevented or treated by administering a compound to a group of cells having an aberration in a signal transduction pathway to an organism. The effect of administering a compound on organism function can then be monitored. The organism is preferably a mouse, rat, rabbit, guinea pig, or goat, more preferably a monkey or ape, and most preferably a human.
In an eleventh aspect, the invention features methods for detection of a kinase polypeptide in a sample as a diagnostic tool for diseases or disorders, wherein the method comprises the steps of: (a) contacting the sample with a nucleic acid probe which hybridizes under hybridization assay conditions to a nucleic acid target region of a kinase polypeptide selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5, said probe comprising the nucleic acid sequence encoding the polypeptide, fragments thereof, and the complements of the sequences and fragments; and (b) detecting the presence or amount of the probe:target region hybrid as an indication of the disease.
In preferred embodiments of the invention, the disease or disorder is selected from the group consisting of rheumatoid arthritis, artherosclerosis, autoimmune disorders, organ transplantation, myocardial infarction, cardiomyopathies, stroke, renal failure, oxidative stress-related neurodegenerative disorders, and cancer. In other preferred embodiments, the kinase polypeptide is selected from the group consisting of PAK4 and PAK5, or the polypeptide is selected from the group consisting of ZC1, ZC2, ZC3, and ZC4, and the disease is cancer.
The kinase xe2x80x9ctarget regionxe2x80x9d is the nucleotide base sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, or SEQ ID NO:106, or the corresponding full-length sequences, a functional derivative thereof, or a fragment thereof to which the nucleic acid probe will specifically hybridize. Specific hybridization indicates that in the presence of other nucleic acids the probe only hybridizes detectably with the kinase of the invention""s target region. Putative target regions can be identified by methods well known in the art consisting of alignment and comparison of the most closely related sequences in the database.
In preferred embodiments the nucleic acid probe hybridizes to a kinase target region encoding at least 6, 12, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids of the sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, or the corresponding full-length amino acid sequence, or a functional derivative thereof. Hybridization conditions should be such that hybridization occurs only with the kinase genes in the presence of other nucleic acid molecules. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 20 contiguous nucleotides. Such conditions are defined supra.
The diseases for which detection of kinase genes in a sample could be diagnostic include diseases in which kinase nucleic acid (DNA and/or RNA) is amplified in comparison to normal cells. By xe2x80x9camplificationxe2x80x9d is meant increased numbers of kinase DNA or RNA in a cell compared with normal cells. In normal cells, kinases are typically found as single copy genes. In selected diseases, the chromosomal location of the kinase genes may be amplified, resulting in multiple copies of the gene, or amplification. Gene amplification can lead to amplification of kinase RNA, or kinase RNA can be amplified in the absence of kinase DNA amplification.
xe2x80x9cAmplificationxe2x80x9d as it refers to RNA can be the detectable presence of kinase RNA in cells, since in some normal cells there is no basal expression of kinase RNA. In other normal cells, a basal level of expression of kinase exists, therefore in these cases amplification is the detection of at least 1-2-fold, and preferably more, kinase RNA, compared to the basal level.
The diseases that could be diagnosed by detection of kinase nucleic acid in a sample preferably include cancers. The test samples suitable for nucleic acid probing methods of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids. The samples used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample that is compatible with the method utilized.
In a final aspect, the invention features a method for detection of a kinase polypeptide in a sample as a diagnostic tool for a disease or disorder, wherein the method comprises: (a) comparing a nucleic acid target region encoding the kinase polypeptide in a sample, where the kinase polypeptide is selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5, or one or more fragments thereof, with a control nucleic acid target region encoding the kinase polypeptide, or one or more fragments thereof; and (b) detecting differences in sequence or amount between the target region and the control target region, as an indication of the disease or disorder. Preferably, the disease or disorder is selected from the group consisting of immune-related diseases and disorders, organ transplantation, myocardial infarction, cardiovascular disease, stroke, renal failure, oxidative stress-related neurodegenerative disorders, and cancer. Immune-related diseases and disorders include, but are not limited to, those discussed previously.
The term xe2x80x9ccomparingxe2x80x9d as used herein refers to identifying discrepancies between the nucleic acid target region isolated from a sample, and the control nucleic acid target region. The discrepancies can be in the nucleotide sequences, e.g. insertions, deletions, or point mutations, or in the amount of a given nucleotide sequence. Methods to determine these discrepancies in sequences are well-known to one of ordinary skill in the art. The xe2x80x9ccontrolxe2x80x9d nucleic acid target region refers to the sequence or amount of the sequence found in normal cells, e.g. cells that are not diseased as discussed previously.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. For example, in some instances the nucleotide sequence of the ZC4 kinase polypeptide may not be part of a preferred embodiment.
The summary of the invention described above is not limiting and other features and advantages of the invention will be apparent from the following detailed description of the invention, and from the claims.